Characterization of Active Mitogen-Activated Protein Kinase in Ovarian Serous Carcinomas

Ovarian cancer is the leading cause of death in women who suffer from gynecologic cancer. Of the various histologic subtypes of ovarian cancer, ovarian serous carcinoma is the most common and corresponds to what is generally referred to as ovarian cancer. Based on a number of clinicopathologic and molecular genetic studies (1), we have recently proposed a dualistic model for the development of ovarian serous carcinoma (2, 3). In this model serous carcinomas are subdivided into low- and high-grade tumors. Low-grade serous carcinoma, also known as invasive micropapillary serous carcinoma, develops in a step-wise fashion from an intraepithelial (in situ) low-grade serous carcinoma, which in turn arises from an atypical proliferative serous tumor. These two noninvasive neoplasms correspond to borderline tumors. Atypical proliferative serous tumor may develop either from a cystadenoma or directly from ovarian surface epithelium. High-grade serous carcinoma develops in a de novo fashion directly from ovarian surface epithelium or from small epithelial inclusion cysts as no intermediate stages have thus far been identified. Low-grade serous carcinomas typically have an indolent course with recurrences developing over many years, but eventually these patients develop intra-abdominal carcinomatosis, which proves to be fatal. High-grade serous carcinomas are highly aggressive, and most patients present with advanced stage disease at the time of diagnosis (4). Recent studies have shown that activating mutations of BRAF or KRAS are found in ∼60% of serous borderline tumors (atypical proliferative tumors and intraepithelial low-grade serous carcinomas) and low-grade serous carcinomas but rarely in the high-grade serous carcinomas (1). This suggests that the signaling pathway involving KRAS and BRAF is important in the development of low-grade serous tumors.

The mitogen-activated protein kinase (MAPK), also known as extracellular signal-regulated protein kinase (ERK), is a downstream target of the RAS, RAF, and MAP/ERK kinase and is crucial for transduction of growth signals from several key growth factors, cytokines, and proto-oncogenes (5). Mutations or overexpression of components, including KRAS and BRAF, in the MAPK pathway lead to constitutive activation of MAPK by phosphorylation. Activation of MAPK in turn activates downstream protein kinases, nuclear proteins, and transcription factors (6), which may contribute to the tumor development (7). Besides its role in oncogenesis, activation of MAPK may sensitize cancer cells to anticancer agents (5) as activation of MAPK is essential for the killing effect of CD437, a synthetic retinoid that has been reported to induce growth arrest and apoptosis in ovarian cancer cell lines (8). It has been shown that constitutive activation of the MAPK signaling pathway occurs in a wide variety of human carcinomas including pancreatic, colorectal, lung, breast, and prostate carcinomas (9). In this study, we attempted to characterize the expression of active (phosphorylated) MAPK in ovarian serous tumors by assessing its differential expression in low-grade and high-grade serous tumors. In addition, we examined whether phosphorylated MAPK alone, or in combination with other clinical parameters, could serve as a prognostic marker.

Formalin-fixed, paraffin-embedded tissue samples of 207 ovarian serous tumors including 29 serous cysts, 7 atypical proliferative serous tumors, 15 intraepithelial low-grade serous carcinomas, 16 low-grade serous carcinomas, and 140 high-grade serous carcinomas were retrieved from the archival file of the Department of Pathology from the Johns Hopkins Hospital. The acquisition of paraffin tissues was approved by the Johns Hopkins Institutional Review Board. The paraffin tissues were organized into tissue microarrays, which were made by removing three 1.5-mm diameter cores of tumor from each block. The selection of areas for core was made by two pathologists (Chih-Yi Hsu and Ie-Ming Shih) based on review of the H&E slides. To evaluate whether expression of active MAPK was a prognostic marker in patients with ovarian serous carcinoma, we analyzed 117 advanced stage high-grade serous carcinomas of which 96 were Fédération Internationale des Gynaecologistes et Obstetristes (FIGO) stage III and 21 were FIGO stage IV.

Expression of the active (phosphorylated) form of MAPK was assessed by immunohistochemistry and Western blot analysis. The antibody used was a rabbit polyclonal antibody, pTEpY, that specifically reacts with the phosphorylated but not the unphosphorylated MAPK (Promega, Madison, WI). Immunohistochemistry was done on tissue microarrays at a dilution of 1:500 followed by the EnVision+System with the peroxidase method (DAKO, Carpinteria, CA). The percentage of positive cells was estimated by randomly counting ∼500 tumor cells from three different high-power fields (×40) within one specimen. For the negative control, an isotype (IgG1) matched antibody, MN-4, was used in parallel (10). A positive reaction was defined as discrete localization of the brown chromagen in the nucleus or cytoplasm. Cases in which more than 5% of the tumor cells showed detectable immunoreactivity were scored as positive.

Western blot analysis was done with the same antibody (1:3,000) on 2 representative ovarian serous carcinomas and one specimen of an ovarian surface epithelial cell culture. One of the ovarian serous carcinoma cell cultures was previously treated with CI-1040, a compound that specifically inhibits the activity of MEK which activates (phosphorylates) MAPK (7). For positive control, the same blot was incubated with an antibody that reacts to glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Research Diagnostics, Inc., Flanders, NJ) at a dilution of 1:2,000. Similar amounts of total protein from each lysate were loaded and separated on 12% Tris-glycine-SDS polyacrylamide gels (Novex, San Diego, CA) and electroblotted to Millipore Immobilon-P polyvinylidene difluoride membranes. Western blots were developed by chemiluminescence (Pierce, Rockford, IL).

Drug sensitivity was assessed by an agar-based cell culture system with radioactive thymidine incorporation as the end point (Oncotech, Tustin, CA). There were 59 cases in which the results of this assay were available for study. The protocol of the assay has been detailed online.⁵ Briefly, fresh tumor specimens immediately removed at the time of surgery were placed into the RPMI (Life Technologies, Inc., Carlsbad, CA) and shipped to Oncotech, Inc. for analysis. The tumor tissue was minced and enzymatically digested to disaggregate the tumor cells, which were then plated in soft agar. Tumor cells were then exposed to antineoplastic agents at a concentration of a maximum tolerated dose for 5 days in vitro. Tritiated thymidine was introduced during the last 2 days of culture as a measure of cell proliferation, and the drug treated cells were compared with the untreated controls. Assay results were divided into three categories: extreme, intermediate, and low drug resistance with the criteria described in the company’s web site. The extreme drug resistance was interpreted as drug resistant, and the intermediate and low drug resistance were interpreted as drug sensitive in this study as reported previously (11, 12).

For nucleotide sequencing, genomic DNA was purified from microdissected tumors as described previously (1). The mutational analysis was done with the software of Lasergene (DNAstar, Madison, WI).

The significance of age for high-grade versus low-grade ovarian serous tumors was tested by the Mann-Whitney test. The significance of expression of active MAPK and patient age, FIGO stage, and in vitro drug resistance was determined using the χ² or the Fisher’s exact test. Overall survival was defined as the time between diagnosis and death or the most recent follow-up. The survival curves were established using the Kaplan-Meier method, and their differences were tested by the log-rank test. All of the patients with advanced high-grade serous carcinomas were treated with a combined chemotherapy including paclitaxel, carboplatin, or cisplatin.

Patients with high-grade serous carcinoma [median (range), 61 (33 to 86) years] were older than patients with low-grade serous carcinoma [39 (24 to 65) years; Mann-Whitney test, P < 0.001]. Expression of the active MAPK in ovarian serous tumors was assessed with immunohistochemistry. The specificity of the antibody for binding with the active (phosphorylated) form of MAPK was show by Western blot analysis. As shown in Fig. 1, a single band of M_r 42,000 corresponding to the phosphorylated MAPK is present in both the high-grade and low-grade serous carcinoma cell cultures but not in ovarian surface epithelial cells. The band from the low-grade tumor cells was not detectable in cells pretreated with CI-1040 that inhibited phosphorylation of MAPK, confirming the specificity of the antibody.

Because of the frequent mutations in KRAS and BRAF in ovarian low-grade serous tumors as compared with high-grade serous carcinoma (1, 13), we examined whether there is a differential expression of activated MAPK in low-grade and high-grade serous tumors. The immunoreactivity of active (phosphorylated) MAPK was detected in both the nucleus and the cytoplasm of the tumor cells (Fig. 2), which is consistent with a previous report (14). The frequency of expression of active MAPK in high-grade serous carcinomas (41%) was significantly lower than in low-grade serous carcinomas (81%), intraepithelial low-grade serous carcinomas (80%), and atypical proliferative serous tumors (71%; χ² test, P < 0.001; Table 1). Among the high-grade serous carcinomas, the expression of active MAPK was more frequent in younger patients (53% in patients <60 years versus 31% in patients ≥60 years old; χ² test, P = 0.010). Expression of active MAPK did not correlate with FIGO stage (P = 0.398) nor with drug resistance for paclitaxel (P = 0.893), cisplatin (P = 0.071), and carboplatin (P = 1.000) that are commonly used in treating ovarian cancer patients (Table 2). In low-grade serous tumors, expression of activated MAPK was independent of age (Mann-Whitney test, P = 0.368) and FIGO stage (χ² test, P = 0.777).

To assess if there was a correlation of expression of active MAPK and mutation in KRAS or BRAF, we compared the immunoreactivity of active MAPK in 28 cases of low-grade serous carcinomas and 25 cases of high-grade serous carcinomas that were available for mutational analysis (Table 3). Mutational status of KRAS and BRAF in 38 of the 53 cases has been reported in our previous study (1). In low-grade serous tumors, all of the 19 cases with mutant KRAS or BRAF were MAPK positive, whereas 5 of 9 low-grade tumors with wild-type KRAS and BRAF were MAPK positive. Thus, mutations in KRAS or BRAF correlated with the immunoreactivity of active MAPK (100 versus 55.6%; Fisher’s exact test, P = 0.006). In high-grade serous carcinomas, all of the 25 cases contained wild-type KRAS and BRAF. Nine of the high-grade serous carcinomas were positive for activated MAPK.

Next, we evaluated whether expression of active MAPK was a prognostic marker in patients with ovarian serous carcinoma. We focused on 117 advanced stage high-grade serous carcinomas because this is the most common clinical presentation in patients with ovarian cancer. As compared with high-grade serous carcinomas, low-grade serous carcinomas are rare, and the case number is not sufficient for statistical analysis of cumulative survival. The median survival for stage III patients was 51 months, and the median survival for stage IV patients was only 30 months (log-rank test, P = 0.002). The 2-year and 5-year survival rates in patients whose tumors expressed active MAPK appeared better than those not expressing it (90.4 versus 71.1% and 58.6 versus 37.3%, respectively) but was statistically significant only for the 2-year survival (log-rank test, P = 0.037; Fig. 3,A). Patient age (log-rank test, P = 0.490) and the status of in vitro drug resistance (paclitaxel P = 0.232, cisplatin P = 0.151, and carboplatin P = 0.323) did not reveal a significant difference in the survival for advanced stage high-grade serous carcinoma (Fig. 3, B–D) but was significantly better for survival when tumors expressed active MAPK and were sensitive to paclitaxel (5-year survival 74.9 versus 31.0%; log rank test, P = 0.048; with age adjusted, P = 0.014; Fig. 3,E). In contrast, expression of active MAPK failed to show such significant prognostic difference in combination with cisplatin and carboplatin. When the patients at advanced stages were stratified into stage III and stage IV groups, 33 patients at stage III with active MAPK expression and paclitaxel sensitivity had even better overall survival with a 5-year survival of 87.5 versus 39.2% in MAPK-positive cases (log rank test, P = 0.043; with age adjusted, P = 0.027; Fig. 3 F). It lacked a statistical power to assess the significance in stage IV patients because of a limited sample size (n = 10).

In this study, active MAPK was more frequently expressed in low-grade as compared with high-grade ovarian serous carcinomas. This finding is consistent with molecular genetic studies showing that activating mutations in KRAS and BRAF, the upstream regulators of MAPK, are common (>60%) in low-grade carcinomas and their precursors, serous borderline tumors (atypical proliferative serous tumor and intraepithelial low-grade serous carcinomas) but rare in high-grade serous carcinomas (1) and provides additional support for a new model of ovarian carcinogenesis (2). Expression of active MAPK correlated with mutations in KRAS or BRAF but was also detected in some low-grade tumors and high-grade carcinomas with wild-type KRAS and BRAF, suggesting that activation of MAPK could be because of other events besides mutation of KRAS and BRAF such as overexpression of genes upstream to MAPK. Therefore, this study provides additional support to our hypothesis of dual pathways for ovarian carcinogenesis (1, 2).

Analysis of MAPK expression with clinical outcome revealed that among the patients with stage III and IV high-grade serous carcinoma, expression of active MAPK was associated with better overall survival—a finding that was previously reported with ascites samples of ovarian serous carcinomas (15). Furthermore, our finding showed a good prognosis in patients whose tumors were sensitive to paclitaxel and expressed active MAPK. This finding contrasts to breast carcinoma in which expression of active MAPK is a poor prognostic marker (16). This difference in ovarian and breast carcinoma is intriguing and probably reflects different roles of the MAPK signaling pathway in different tumor types (5). Recent studies have shown that MAPK activation is necessary for cancer cell death initiated by a variety of anticancer agents (5, 8). For example, activation of MAPK is essential for the killing effect of CD437, a synthetic retinoid that has been reported to induce growth arrest and apoptosis in ovarian cancer cell lines (8). Additional studies are necessary to determine whether constitutive expression of active MAPK sensitizes ovarian cancer cells to paclitaxel in vivo.

In conclusion, the results of this study showing a differential expression of active MAPK in low-grade serous carcinoma compared with high-grade serous carcinoma together with previous molecular genetic findings of frequent mutations of KRAS and BRAF in low-grade serous tumors support the view that low-grade and high-grade serous tumors develop along different pathways. Active MAPK in combination with in vitro paclitaxel sensitivity may serve as a prognostic marker in patients with advanced stage high-grade serous carcinoma. This finding would have important implications for clinical management of ovarian cancer patients.

We thank the immunohistochemistry laboratory for excellent immunostaining and the members in the Molecular Genetics of Ovarian Cancer Laboratory at Johns Hopkins Medical Institutions for previewing the manuscript.